[0001] The present invention relates to a hybrid gene containing a portion of the HIV-1
envelope gene and a portion of the HTLV-I envelope gene. The invention also relates
to the corresponding gene products, the recombinant vectors encoding said gene products,
unicellular hosts containing these recombinant vectors, methods for producing said
gene products by using unicellular hosts containing the the recombinant vectors, and
methods for detecting the presence of antibodies to HIV-1 and HTLV-I and HTLV-II envelope
gene products in mammalian body fluids.
BACKGROUND OF THE INVENTION
[0002] Human T Cell Lymphotropic Virus Type 1 (HTLV-I) is a Type C RNA virus which has been
identified as the cause of Adult T-Cell Leukemia/Lymphoma (ATL). HTLV-I was the first
retrovirus to be isolated and cloned from patients with ATL, and has also been linked
to neurological diseases such as tropical spastic paraparesis and HTLV-I associated
myelopathy. In certain areas of the world HTLV-I infections appear to be endemic.
For example, in certain Southern Japanese populations the seroprevalence of HTLV-I
is known to be as high as 35%. While the frequency of HTLV-I infection in the U.S.
is still quite low, infection by HTLV-I alone or in combination with other human retroviruses
appears to be a growing problem in the inner-city populations where intravenous drug
abuse is a common practice. Another isolate, designated HTLV-II, derived from a patient
with T-cell hairy cell Leukemia is closely related to HTLV-I.
[0003] Once the complete nucleotide sequence of the HTLV-I genome was determined, manufacture
of the corresponding proteins followed, further enabling the detection of antibodies
to these proteins. When techniques such as Western Blot and radioimmune precipitation
(RIP) were adapted to measure human antibodies to HTLV-1 core and elvelope proteins,
it became evident that HTLV-1 infection was nearly endemic in certain populations.
It was also found that antibodies from a patient with hairy cell leukemia, whose disease
was associated with HTLV-II, strongly reacted to a polypeptide corresponding with
the p21 region of the HTLV-I envelope indicating the high degree of homology between
HTLV-I and HTLV-II.
[0004] However, the importance of HTLV-I as a human pathogen has not been recognized because
the AIDS epidemic has consumed the interest of researchers and clinicians. AIDS is
due to infection by the HIV-1 retrovirus. Both HIV-1 and HTLV-I are transmitted in
a variety of ways, which include contaminated needles, blood transfusions, sexual
contact and vertical transmission from mother to offspring. Since both viruses are
associated with human disease, investigators have now begun to screen cross-sections
of the American population for antibodies to HTLV-I as well as HIV-1.
[0005] At present there are separate screening tests for antibodies to HTLV-I and HIV-1,
both of which use disrupted virions obtained from virally transformed human T cell
lines. The viral antigens are semipurified and coated onto a solid phase which facilitates
binding of human antibodies, if present, to the viral antigens. The currently available
HTLV-I assays exhibit inherent difficulties. Since HTLV-I retroviruses bud from cell
membranes, there will be human cellular membrane determinants present in the viral
envelope. The cross-reactivity of endogenous autoimmune antibody with these determinants
leads to a substantial number of false positives. Falsely negative values are also
common because the immunoreactive viral envelope protein is often destroyed during
the purification of the virus prior to its use in the screening assay. The same problems
are inherent in the currently available HIV-1 screening assays.
[0006] Expression of recombinant HTLV-I and HIV-1 gene products in bacterial and yeast systems
has now been reported and peptides corresponding to specific genes have been isolated.
These recombinantly produced peptide antigens are cross-reactive with human sera from
patients who are known to be seropositive for HTLV-I and HIV-1. Separate antibody
screening assays which utilize recombinantly expressed protein antigens are currently
being developed for both HTLV-I and HIV-1.
SUMMARY OF THE INVENTION
[0007] The present invention provides hybrid genes comprising a first DNA sequence coding
for a polypeptide containing at least one epitope of the HIV-1 envelope protein which
is recognized by antibodies to HIV-1, and a second DNA sequence coding for a polypeptide
containing at least one epitope of the HTLV-I envelope protein which is recognized
by antibodies to HTLV-I. This hybrid gene is useful for the production of the corresponding
hybrid protein, a source of DNA for other purposes, or other related purposes.
[0008] There are two preferred embodiments of the genes. First is a gene in which the first
DNA sequence codes for a polypeptide containing the immunodominant region of the HIV-1
gp41 transmembrane protein, preferably approximately amino acids 560-620 of the HIV-1
envelope protein (numbered according to Figure 5), and the second DNA sequence codes
for a polypeptide containing approximately amino acids 306-440 of the HTLV-I envelope
protein.
[0009] The second preferred embodiment is a gene in which the first DNA sequence codes for
a polypeptide containing the immunodominant region of the HIV-1 gp41 transmembrane
protein, preferably approximately amino acids 560-620 of the HIV-1 envelope protein
and the second DNA sequence codes for a polypeptide containing approximately amino
acids 201-308 of the HTLV-I envelope protein.
[0010] The present invention also provides the corresponding hybrid proteins comprising
a first amino acid subsequence containing at least one epitope of the HIV-1 envelope
protein which is recognized by antibodies to HIV-1, and a second amino acid subsequence
containing at least one epitope of the HTLV-I envelope protein which is recognized
by antibodies to HTLV-I. The hybrid proteins of the present invention are useful as
antigens to detect the presence of antibodies to the HTLV-I and HlV-1 envelope proteins,
as immunogens, as well as other therapeutic or diagnostic purposes.
[0011] There are two preferred embodiments of the polypeptides. First is a polypeptide where
the first amino acid subsequence contains the immunodominant region of the HIV-1 gp41
transmembrane protein, preferably approximately amino acids 560-620 of the HIV-1 envelope
protein (said amino acids numbered in accordance with Figure 5) and the second amino
acid subsequence contains approximately amino acids 306-440 of the HTLV-I envelope
protein.
[0012] Second is a polypeptide where the first amino acid subsequence contains the immunodominant
region of the HIV-1 gp41 transmembrane protein, preferably approximately amino acids
560-620 of the HIV-1 envelope protein and the second amino acid subsequence contains
approximately amino acids 201-308 of the HTLV-I envelope protein.
[0013] The present invention also comprises recombinant vectors comprising the hybrid genes
of the present invention, particularly a recombinant vector which is a plasmid capable
of replication in a unicellular host.
[0014] The present invention also comprises unicellular hosts carrying the recombinant vectors
of the present invention, as well as a method for producing the hybrid proteins of
the present invention by culturing a unicellular host containing a recombinant vector
of the present invention under appropriate conditions of growth to produce the corresponding
hybrid proteins.
[0015] The present invention also provides a method for detecting antibodies to HTLV-I envelope
proteins, HTLV-II envelope proteins and/or HIV-1 envelope proteins in mammalian body
fluids comprising contacting a test sample containing antibodies with a hybrid protein
containing at least one epitope of the HIV-1 envelope protein and at least one epitope
of the HTLV-I envelope protein and allowing protein-antibody complexes to form and
detecting the complexes, thereby demonstrating the presence of antibodies in the sample.
Preferred is where the method is an immunoassay such as Western Blotting, Double Antigen
Sandwich Method, Radioimmunoassay or, particularly, Enzyme Immunoassay.
[0016] The present invention also comprises a diagnostic test kit useful for the detection
of antibodies to HIV-1, and/or HTLV-I, or HTLV-II in mammalian body fluids.
[0017] Both HTLV-I and HIV-1 are very important human pathogens so a single screening test
which measures antibodies to both HTLV-1 and HIV-1 is highly desirable.
DESCRIPTION OF DRAWINGS
[0018]
Fig. 1 Illustrates the construction of plasmids HIV-1 pENV(60)-HTLV-I-ENV-1 and -ENV-2 pENV(60)-HTLV-I-ENV1
and-ENV-2. The HTLV-I genome and restriction enzyme fragments of the envelope gene
used for subcloning are shown. (For details see Examples 1 and 2). The cloning and
expression of HIV-1 pENV(80) has been described by Certa et al., EMBO J. 5; 3051-3056
(1986).
Fig. 2 The HIV-1-HTLV-I translation product of plasmid HIV-1 pENV(60)-HTLV-I-ENV-1 is set
forth immediately prior to its complete DNA and amino acid sequence (single letter
code designations for amino acids indicate residues which are vector-related and not
specific to the envelope genes).
Fig. 3 The HIV-1-HTLV-I translation product of plasmid HIV-1 pENV(60)-HTLV-I-ENV-2 is set
forth immediately prior to its complete DNA and amino acid sequence (single letter
code designations for amino acids indicate residues which are vector-related and are
not specific to the envelope genes).
Fig. 4 Illustrates the expression of HIV-1 pENV(60)-HTLV-I-ENV-1 and -ENV-2 fusion proteins
and resolution of induced products on SDS-PAGE. Samples were prepared as described
in Example 5 and visualized in gels with Coomassie blue staining. The contents of
the lanes shown are as follows: E. coli W 3110 cells containing pDMI,1 and HIV-1 pENV(60)-HTLV-I-ENV-1
uninduced (lane 1), 4 hour induced (lane 2) and Sephacryl S-200 purified (lane 3),
E. coli Je 5506 cells containing pDMI,1 and HIV-1 pENV(60)-HTLV-I-ENV-2 uninduced
(lane 4), 4 hour induced (lane 5), and Sephacryl S-200 purified (lane 6). M represents
molecular weight standards.
Fig. 5 Illustrates the amino acid sequence of the HIV-1 envelope protein.
DETAILED DESCRIPTION
[0019] Preferred is a hybrid gene having the DNA sequence:

[0020] Also preferred is a hybrid gene having the DNA sequence:

[0021] As shown above, the hybrid genes of the present invention may additionally contain
"initiation" and "termination" DNA sequences. These sequences arise as a result of
the recombinant vector and are essential for expression of the hybrid genes in the
unicellular host. The initiation sequences are operatively linked to the hybrid genes.
Various initiation sequences, all of which may exhibit different nucleotide sequences,
may be used with the present hybrid genes and the invention is intended to include
the hybrid genes in combination with all these sequences. The preferred initiation
DNA sequence of the present invention has the nucleotide sequence:
ATG AGA GGA TCC
[0022] The "termination" DNA sequences which may additionally be present after the terminal
codon of the hybrid genes also result from the recombinant vectors used for expression
of the corresponding gene products. The termination DNA sequences will vary with the
recombinant vectors used. The present invention is intended to include all the various
termination DNA sequences.
[0023] In the hybrid genes of the present invention the two DNA sequences may be fused directly
to one another or they may be attached to one another via "linker" sequences. A linker
sequence may be inserted between the HIV-1 envelope gene sequence and the HTLV-I envelope
gene sequence to maintain the proper reading frame during translation of the hybrid
proteins. The present invention is intended to include the various linker DNA sequences
which may be used for this purpose.
[0024] The preferred hybrid genes of the present invention code for the polypeptides having
the amino acid sequences of the formulae:

and

[0025] These polypeptides may additionally contain sequences of several amino acids which
are coded for by the corresponding "termination" or "linker" DNA sequences mentioned
previously. The present invention is intended to include the various amino acid sequences
coded for by the "termination" or "linker" sequences, none of which affect the functionality
of the hybrid polypeptides but occur as a result of expression in the transformed
host.
[0026] The hybrid proteins themselves may also contain amino acid substitutions if such
substitutions do not generally alter the biological activity of the hybrid proteins.
Amino acid substitutions in polypeptides which do not essentially alter their biological
activities are know in the art and described, e.g. by H. Neurath and R.L. Hill in
"The Proteins", Academic Press, New York (1979). The most frequently observed amino
acid substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly,
and vice versa.
[0027] Further, given the similarity of the HTLV-I and HTLV-II viruses, antibodies to HTLV-II
will also recognize the present hybrid polypeptides. The present invention is also
intended to include the detection of antibodies to HTLV-II in mammalian body fluids
according to the invention. [see European Patent Application, Publication No. 181,107].
The hybrid genes and their preparation
[0028] The hybrid genes of the present invention are made by fusing a DNA sequence containing
at least one epitope of the HTLV-I envelope protein which recognizes antibodies to
HTLV-I to a DNA sequence containing at least one epitope of the HIV-1 envelope protein
which recognizes antibodies to HIV-1.
[0029] The HTLV-I and HIV-1 envelope gene fragments can be obtained from a variety of sources.
[0030] The HTLV-I envelope gene codes for a glycoprotein of 61,000 daltons (gp61) which
is cleaved into a 46,000 M.W. exterior glycoprotein (gp46) and the 21,000 M.W. transmembrane
protein (gp21).
[0031] HTLV-I envelope gene fragments which can be used for the manufacture of the hybrid
genes are available from a number of sources. For example, in the present invention
a plasmid designated pH2Ex was used as a source of HTLV-I envelope gene DNA. This
plasmid was originally subcloned from a plasmid designated λCR1 which contains Env,
pX, and LTR of HTLV-I as described in Manzari et al., PNAS 80, 1574-1578 (1983) and
European Patent Application, Publication No. 181 107.
[0032] Plasmid pKS300, which codes for part of the C-terminal region of gp46 of the HTLV-I
envelope proteins, and plasmid pKS400, which codes for part of the N-terminal region
of gp21 of the HTLV-1 envelope protein can also be used and may be easily derived
from the above mentioned plasmid pH2Ex by the skilled artisan utilizing known methods.
These plasmids are the subject of European Patent Application, Publication No. 181
107.
[0033] Other possible sources of HTLV-I envelope gene fragments include chemical synthesis
of the gene sequences according to methods known in the art [Certa et al., EMBO J,
5, 3051-3056 (1986)].
[0034] HTLV-I envelope gene fragments can also be obtained from human T-cell lines which
have been virally transformed by HTLV-I virions as set forth in Yoshida et al., PNAS
79, 2031-2035 (1982) and Poiesz et al., PNAS 77, 7415-7419 (1980). The DNA fragments
can then be isolated by methods known in the art, a cDNA library constructed, and
the desired envelope gene fragments can be obtained by probing the cDNA library.
[0035] The HIV-1 envelope gene codes for a glycosylated protein (gp) with a molecular weight
of 160,000 (gp160) that is processed to gp120 and gp41. HIV-1 envelope gene fragments
which can be used to make the fusion gene are widely available. The HIV-1 envelope
gene sequence desired can be chemically synthesized according to known methods, or
the DNA sequences can be isolated by preparing DNA which is complementary to the mRNA
produced from the virally transformed cell lines. The HIV-1 virus has been successfully
cultured using the H-9 cell line [PCT application, Publication No. WO 85/04897] and
this can be one source of HIV-1 envelope DNA fragments. The entire genome of HIV-1
has been molecularly cloned as set forth in Schüpbach et al., Science 224,503-505
(1984). The complete nucleotide sequence of the genome has been determined as set
forth in Ratner et al., Nature 313,277-284 (1985). The particular HIV-1 envelope gene
fragment used to construct the hybrid protein of the instant invention was obtained
as described in Certa et al., EMBO J. 5,3051-3056 (1986).
[0036] To construct the hybrid genes of the present invention it is necessary to excise
the desired portions of the HTLV-I envelope gene and the HIV-1 envelope gene utilizing
restriction endonucleases which will "cut" the DNA sequences at specific sites. Examples
1 and 2 illustrate the means by which the hybrid genes are constructed utilizing this
methodology.
[0037] Synthetic hybrid genes can also be manufactured. Oligonucleotide fragments are synthesized
which, when assembled correctly, form a hybrid gene coding for the hybrid protein.
The fragments can be hybridized and ligated in pre-determined stages to construct
the synthetic gene. The synthetic gene can be provided with suitable restriction enzyme
sites and cloned into vectors specifically designed to maximize expression of the
synthetic gene.
[0038] In order to construct said hybrid genes which code for the desired hybrid proteins
several criteria should be observed. Firstly, trinucleotide codons should be used
which are acceptable to or preferably used in the cells, in particular E. coli. Secondly,
it would be desirable to have restriction enzyme recognition sites at the termini
of the molecule so as to allow insertion into a vector of plasmid or phage origin.
Moreover, restriction sites should be selected which allow the use of well-understood
cloning and expression vectors, such as plasmids of the pDS-family. Thirdly, a series
of restriction endonuclease recognition sites should be introduces to exchange and
modify parts of the gene very easily and to allow the expression or certain parts
of it. Fourthly, the synthesis should not be unnecessarily complicated and illegitimate
cross-hybridizations should be minimized in order to facilitate gene assembly.
[0039] The hybrid genes of the present invention can be introduced in any convenient expression
vector of plasmid or phage origin in a manner known per se. Convenient expression
vectors of plasmid or phage origin are mentioned e.g., in the laboratory manual "Molecular
Cloning" by Maniatis et al., Cold Spring Harbor Laboratory, 1982.
The recombinant vectors and transformed hosts
[0040] Once the hybrid genes have been constructed they can be introduced into any convenient
expression vector, plasmid or phage in any manner known per se. Convenient recombinant
vectors of plasmid or phage origin are mentioned, for example, in the Laboratory Manual
"Molecular Cloning", Maniatis et al., Cold Spring Harbor Laboratory, 1982.
[0041] E. coli strains containing plasmids useful for these constructions are, e.g. E.coli
M15 containing plasmids pDS5/RBSII,3A+5A and pDMI,1 (DSM 3517), E. coli M15 containing
plasmids pDS6/RBSII,3A+5A and pDMI,1 (DSM 3518) or E. coli TB1 containing plasmid
pA-ENV-20 (DSM 3516). These strains are all freely available upon request from Deutsche
Sammlung von Mikroorganismen (DSM) in Göttingen.
[0042] As discussed previously, the hybrid genes, when inserted into the appropriate site
of the recombinant vectors, may be fused to DNA sequences which are not part of the
structural genes themselves. It is only required, however, that the inserted gene
sequences, in the transformed hosts, produce hybrid proteins or analogs obtained by
amino acid substitutions.
[0043] Methods for expressing DNA sequences which code for the hybrid proteins of the present
invention are well known, e.g. from Maniatis et al, supra. They include transforming
an appropriate host with a recombinant vector having the said DNA sequence operatively
linked to an expression control DNA sequence, culturing the transformed host under
appropriate conditions of growth and extracting and isolating the desired hybrid protein
from the culture. The skilled artisan may select from these known methods those which
are most effective for a particular gene expression without departing from the scope
of the present invention.
[0044] The selection of a particular host for use in this invention is dependent upon a
number of factors recognized by the art. These include, for example, compatibility
with the chosen expression vector, toxicity of the proteins encoded for the hybrid
plasmid, ease of recovery of the desired protein, expression characteristics, biosafety
and costs. Within these general guidelines, examples of useful bacterial hosts are
gram-negative and gram-positive bacteria, especially strains of E. coli. The most
preferred host cells of the present invention are E. coli M15 (described as DZ291
in M.R. Villarejo et al., J. Bacteriol. 120, 466-474 [1974]) and E. coli Je 5506 (described
in Hirota et al., PNAS 74, 1417-1420 [1977]). However, other E. coli strains, such
as E. coli 294 (ATCC No. 31446), E. coli RR1 (ATCC No. 31343) and E. coli W3110 (ATCC
No. 27325), can also be used.
[0045] As produced in E. coli, the hybrid proteins of the present invention are largely
confined to insoluble aggregates of the bacterium, a fact which greatly facilitates
purification of these proteins. To isolate the hybrid proteins of the present invention
the bacterial cells are disrupted or lysed and the insoluble proteins are recovered
by centrifugation. Substantial purification can then be obtained by sequential washing
of the precipitate with buffer, detergent and salt solutions followed by the use of
standard protein purification techniques.
[0046] For small amounts of material such as samples taken for polyacrylamide gel electrophoretic
analysis, the cells can be disrupted by treatment with a detergent such as sodium
dodecyl sulfate (SDS). Larger quantities of the proteins can be recovered by sonication,
or by other mechanically disruptive means, such as the French pressure cell. Preferably,
the cells are lysed by chemical and enzymatic means, such as EDTA or EGTA and lysozyme.
[0047] Preferably the insoluble protein precipitate is washed in three separate steps although
it is foreseeable that certain of these steps could be combined, rearranged or eliminated.
The first step of washing the precipitate may be done with a buffer solution, preferably
10mM Tris, pH 8.0. In the second step the precipitate is washed with a detergent solution,
preferably 1.0% Triton X-100. The third and final wash is with a salt solution, preferably
7M Guanidine-HCl.
[0048] The hybrid protein precipitate resulting from the final wash solution can then be
further purified by conventional protein purification techniques including but not
limited to gel filtration, chromatography, preparative flat-bed iso-electric focusing,
gel-electrophoresis, high performance liquid chromatography (hereinafter HPLC: including
ion exchange, gel filtration and reverse phase chromatography), and affinity chromatography,
e.g. on dye bound carrier or on Sepharose coupled with monoclonal antibodies against
said hybrid protein. Preferably the hybrid protein precipitate is further purified
by size exclusion chromatography using Superose 12 or Sephacryl S-200 gels.
[0049] The methods of this invention entail a number of steps which, in logical sequence,
include (1) preparation of the genes encoding the hybrid gene sequences, (2) insertion
of these genes into appropriate cloning vehicles to produce recombinant vectors containing
said hybrid gene sequences, (3) transfer of the recombinant cloning vehicles into
compatible host organisms, (4) selection and growth of such modified hosts that can
express the inserted gene sequences, (5) identification and purification of the gene
products, (6) use of the gene products to detect antibodies against HIV-1 or HTLV-I
or related viruses.
[0050] The hybrid proteins of the present invention can be produced by conventionally known
methods. The processes by which the novel hybrid proteins can be produced can be divided
into three groups: (1) chemical synthesis, preferably solid-phase synthesis; (2) preparation
of the fusion genes prepared by chemical synthesis which are inserted into a host
and expression of the hybrid proteins by the host; and (3) the corresponding hybrid
genes are obtained from sources such as plasmids or clones as set forth above, and
inserted into a host and the proteins are expressed by the host.
[0051] The hybrid proteins of the present invention can be synthesized by utilizing the
general solid phase methods of Merrifield (Journal of American Chemical Society 85,
2149 [1963]).
[0052] The hybrid proteins can also be produced by recombinant methods by inserting the
hybrid gene sequences into an appropriate recombinant vector, and transforming a host.
The transformant is then grown under appropriate culture conditions to yield the corresponding
hybrid proteins. Example 3, infra, illustrates the expression of the recombinant proteins
of the present invention, which is generally described by Maniatis et al., supra.
Methods for detecting antibodies to HIV-1, HTLV-I or HTLV-II in mammalian subjects
[0053] The hybrid proteins of the present invention can be used to detect the presence of
antibodies to both HIV-1 envelope protein and HTLV-I or related viruses (such as HTLV-II)
envelope proteins in mammalian body fluids such as, serum, tears, semen, vaginal secretions
and saliva. The invention encompasses a diagnostic method for testing mammalian body
fluids for the presence of antibodies to HIV-1, HTLV-I, or related viruses and a test
kit useful in this method.
[0054] One method in which antibodies against the HlV-1, HTLV-I or related viruses (such
as HTLV-II) can be detected is the so-called "Western Blotting" analysis (Tobin et
al., PNAS 76, 4350-4354 (1979)]. According to this technique a hybrid protein of the
present invention is transferred electrophoretically from the SDS-polyacrylamide gel
onto nitrocellulose paper. The nitrocellulose paper is then treated with the test
sample. After washing, the nitrocellulose paper is treated with an anti-human IgG
labeled with peroxidase. The peroxidase is then determined by a suitable substrate,
e.g. with o-phenylenediamine. Radioactive or fluorescent labels may also be used.
[0055] A more convenient technique for the determination of antibodies against the HIV-1
or HTLV-I or HTLV-II virus using a hybrid protein of the present invention is an enzyme-linked
immunosorbant assay (ELISA). Such assay comprises:
(a) immobilizing a hybrid polypeptide of the present invention on a solid support;
(b) contacting a test sample with the immobilized polypeptide of step (a) and allowing
immobilizing protein-antibody complex to form;
(c) washing away unbound material from the complexes of step (b); and
(d) detecting such complexes by the addition of a labeled reagent capable of selectively
detecting human antibodies, such as labeled Staphylococcus aureus protein A or anti-human
IgG, thereby demonstrating the presence of antibodies to HIV-1 envelope protein or
HTLV-1 envelope protein in the sample.
[0056] Suitable solid supports are the inner wall of test vessels (test tube, titer plates
or cuvettes of glass or artificial material) as well as the surface of solid bodies
(rods of glass and artificial material, rods with terminal thickening, rods with terminal
lobes or lamellae). Beads of glass or artificial material are especially suitable
solid phase carriers.
[0057] Useful labels are any detectable functionalities which do not interfere with the
binding of reagent and its binding partner. Numerous labels are known for use in ELISA
assays and other immuno assays such as horseradish peroxidase, radioisotopes such
as ¹⁴C and ¹²⁵I, fluorophores such as rare earth chelates for fluorescein, spin labels
and the like.
[0058] In a preferred embodiment of the present invention an ELISA (EIA) assay is run in
a test kit as described below utilizing the hybrid proteins of the present invention
immobilized on beads. To the coated spheres, patient samples, and positive and negative
controls are added and allowed to incubate at 37°C for 30 minutes. The beads are washed
to remove unbound antibodies and then anti-human IgG coupled to horseradish peroxidase
(HRP) are added. The HRP conjugate is allowed to react for 15 minutes at 37°C and
then the spheres are washed to remove unbound HRP conjugate. HRP substrate, o-phenylenediamine
(OPD), is added to the spheres and allowed to react for 15 minutes at room temperature.
The enzyme reaction is terminated by addition of H₂SO₄ and the resulting yellow-brown
color produced by the HRP is read in a spectrophotometer. The optical density at 492
nm indicates the presence of HRP-conjugate bound to the human IgG which is in turn
bound to the antigen on the plate.
[0059] Antibodies to HIV-1 envelope protein or HTLV-I envelope protein can also be determined
utilizing the hybrid proteins of the present invention with an enzyme immunological
test according to the so-called "Double-Antigen-Sandwich-Method". This method is
based on the work of Maiolini, R.I., as described in Immunological Methods 20, 25-34
(1978). According to this method, the test sample is contacted with a solid support
upon which the hybrid proteins of the present invention, labeled with peroxidase,
are coated. The immunological reaction can then be performed in one or in two steps.
If the immunological reaction is performed in two steps then a washing step is performed
between the two incubations. After the immunological reaction or reactions a washing
step is performed. Thereafter the peroxidase is determined with a substrate, e.g.
with o-phenylene diamine.
[0060] Any positive result which may be obtained by utilizing the method of the present
invention to detect antibodies to HIV-1 envelope or HTLV-I envelope protein in mammalian
body fluids, can be confirmed by separately testing the sample for antibodies to HIV-1
envelope or HTLV-I envelope.
[0061] The present invention will be further described i.n connection with the following
examples which are set forth for the purposes of illustration only.
Example 1
Construction of plasmid HIV-1 pENV(60)-HTLV-I-ENV-1
[0062] Plasmid pH2Ex was used as a source of HTLV-I envelope gene DNA. This plasmid was
originally subcloned from λCR1 which contains Env, pX and LTR of HTLV-I (Manzari et
al., supra). Plasmid pBSENV was constructed by inserting a 715 bp XhoI restriction
fragment into the unique salI site of expression vector pDS56/RBSII (European Patent
Application, Publication No. 282 042)]. The inserted DNA fragment encodes the amino
acids 201-440 of the HTLV-1 envelope gene.
[0063] Plasmid HIV-1 pENV(60)-HTLV I-ENV-1 was constructed by removing the 413 bp BamHI/HindIII
restriction fragment from pBSENV, filling in the recessed 3′-OH ends with the Klenow
fragment of DNA Polymerase I, ligating synthetic HindIII linkers of appropriate size
to maintain the reading frame (New England Biolabs), and inserting this HindIII fragment
into expression vector pDS5/RBSII ENV(60). This expression vector was derived from
clone pDS5/RBSII ENV(80) by removing the HindIII restriction fragment and recircularizing
the remaining vector. This vector now encodes the N-terminal 60 amino acids of a chemically
synthesized DNA fragment which represents an immunodominant region of the HIV 1 gp41
transmembrane protein. Plasmid HIV-1 pENV( 60)-HTLV-I-ENV-1 encodes for a hybrid protein
with a predicted molecular weight of 23.2 Kd containing 60 amino acids of the HIV-1
envelope gene (homologous to amino acids 560-620) and 134 amino acids of the HTLV-I
envelope gene (amino acids 306-440). Additional four amino acids at the N-terminus
are contributed by vector sequences as well as additional thirteen amino acids which
are expressed on the C-terminus of the fusion protein before a termination codon is
recognized (see Fig. 2).
Example 2
Construction of plasmid HIV-1 pENV(60)-HTLV-I-ENV-2.
[0064] Plasmid HIV-1 pENV(60)-HTLV-I-ENV-2 codes for the C-terminus of gp46, the exterior
glycoprotein of HTLV-I (amino acids 201-308). It was constructed by removing a 328
bp BamHI restriction fragment from pBSENV, attaching HindIII linkers which maintain
the reading frame of the pDS expression vector used and inserting the resulting fragment
into the HindIII site of pDS5/RBSII ENV(60) (Fig. 1 and 3).
Example 3
Expression of Recombinant Proteins.
[0065] Expression of the inserted genes was induced by the addition of isopropyl-β-D-thiogalactopyranoside
(IPTG) to actively growing bacterial cultures as described by Certa et al., supra,
with some modifications. Cultures were grown to an OD600 of 0.6-0.7 at 37°C, induced
by addition of IPTG to 0.5 mM, and aliquots removed after 2-4 hours incubation. After
collecting cells by centrifugation, equivalent amounts of whole cell lysate are run
on 12% SDS-PAGE gels and protein bands visualized by staining with Coomassie Brilliant
Blue (R-250) (Figure 4). Whole cell lysate samples were prepared by resuspending 1.5
ml of centrifuged culture fluid in 2x Laemmli sample buffer and boiled.
Example 4
Purification of Recombinant Proteins.
[0066] Purification of the recombinant fusion proteins was achieved through a modification
of the procedure of Manne at al. [PNAS 82, 376 (1985)]. Briefly, IPTG induced cell
pellets were resuspended in PBS/5 mM EDTA/25% sucrose/1% Triton X-100/1 mM DTT, pH
7.5, at 2 ml per gram of cell paste. Lysozyme was added to 1 mg/ml and the suspension
frozen and thawed 3 times. DNase 1 was added to 0.4 mg/ml, incubated on ice for 20
min., and the lysate was centrifuged at 25,000 x g for 15 min. at 4°C. The resulting
pellet was washed 4 times with 25% sucrose/1% Triton X-100/1mM DTT (20 ml). The pellet
was then washed with PBS/1.75M Guanidine HCl/5mM DTT, pH 7.5, and the pellet solubilized
in 3 ml 10mM Tris/5mM DTT/7M Guanidine HCL, pH 8.0. Insoluble materials were removed
by centrifugation. The supernatant was diluted 1:15 with 10 mM Tris/5 mM DTT, incubated
for 10 min. at room temp. and centrifuged at 12,000 x g for 10 min. at 4°C. The pellet
was washed once with 20 ml 10 mM Tris/5 mM DTT, pH 8.0, and slowly dissolved in 125
mM Tris/5 mM DTT/4% SDS/0.02% NaN₃, pH 6.8, at room temperature overnight. In certain
cases, further purification was accomplished by chromatography on a Sephacryl S-200
(Pharmacia) column equilibrated in 25 mM Tris/1 mM EDTA/100 mM NaCl/0.1% SDS/0.02%
NaN₃, pH 8.0. Fractions were analyzed by SDS-PAGE and those containing the protein
of interest were pooled.
Example 5
Testing of Clinical Samples
[0067] Sera tested were obtained from normal blood donors and AIDS and ARC patients who
were previously shown to be seropositive. HTLV-I seropositive samples were obtained
from Japan. Pooled positive control sera for HIV-1 and HTLV-I was obtained from Western
States Plasma.
Procedure for Bead Coating.
Reagents:
[0068]
1. 0.05 M Carbonate - Bicarbonate buffer, pH 9.5.
2. Polystyrene beads (alcohol washed).
3. 1% Tween 20 in 0.05 carbonate-bicarbonate buffer, pH 9.5.
4. HIV-1/HTLV-I fusion protein in 25 mM Tris, 1 mM EDTA, 100 mM NaCl, 0.1% SDS, 0.02%
NaN₃, pH 8.0.
5. Coating Solution: HIV-1 HTLV-I protein is diluted with 0.05 M carbonate-bicarbonate
buffer, pH 9.5 to a final concentration of 10 µg/ml.
Protocol:
[0069]
1. Add 500 beads, alcohol washed (70 gr) to 100 ml of 1% Tween 20 in 0.05 carbonate/bicarbonate
buffer, pH 9.5
2. Incubate at 37°C overnight (18 hrs.). Aspirate solution and wash 5 times with 200
ml of deionized water.
Remove last wash by suction.
3. Cover wet beads with 100 ml of coating solutions containing 10 µg/ml HIV-1/HTLV-I
fusion protein.
4. Incubate for 20 hrs. at 37°C.
5. Remove coating solution by suction.
6. Wash 5 times with 200 ml of deionized water.
7. Remove last wash by suction.
8. Dry beads in tumbler at 37°C for 3 hrs. and store at 2.8°C.
Combination Antibody Screen.
Reagents:
[0070]
1. HIV-1/HTLV-I-ENV protein coated beads: Polystyrene frosted bead, coated with 10
µg/ml HIV-1 pENV(60)-HTLV-I-ENV-1 fusion protein solution.
2. Sample diluent: 20% newborn calf serum, 0.5% Tween 20, 0.01% goat IgG, 0.01% Thimerosal,
5 mM EDTA in 0.02 M phosphate buffered saline, pH 7.0.
3. HIV-1 low positive control: heat inactivated human serum positive for antibody
to HIV, 0.1% azide, 0.01% Thimerosal.
4. HIV-1 high positive control: heat inactivated human serum positive for antibody
to HIV, 0.1% azide, 0.01% Thimerosal.
5. HIV-1/HTLV-I negative control: heat inactivated human serum negative for antibody
to HIV-1 and HTLV-I, 0.1% azide, 0.01% Thimerosal.
6. HTLV-I positive control: heat inactivated human serum positive for antibody to
HTLV-I, 0.1% azide, 0.01% Thimerosal.
7. Conjugate reagent: goat anti-human IgG peroxidase (horseradish) labelled, diluted
1:900 (dilution may vary with titer) in 0.1 M Tris acetate, containing 20% treated
fetal calf serum, 0.04% 4-amino antipyrine, 1% Tween 20 (polysorbate 20), 0.1% Kathon
(v/v), pH 7.3.
8. Substrate buffer: 0.1 M potassium citrate, .02% hydrogen peroxide, 0.01% Kathon,
pH 5.25.
9. OPD tablets: 10 mg o-phenylenediamine/tablet.
10. Stopping reagent: 1N sulfuric acid in deionized water.
Assay Procedure:
[0071]
1. Dispense 10 µl of specimen plus 400 ul of sample diluent (1:41 dilution) into the
reaction tube.
2. Add 1 bead.
3. Incubate using Resa COBAS shaker/incubator for 30 min. at 37°C.
4. Wash tubes in washer using deionized water.
5. Add 250 µl of conjugate reagent.
6. Incubate using Resa COBAS shaker/incubator for 15 min. at 37°C.
7. Approximately 10 minutes before the end of immunological incubation, prepare the
OPD substrate solution by dissolving OPD tablets in substrate buffer (add 1 tablet
per 5 ml of substrate buffer).
8. Wash tubes in the washer using deionized water.
9. Add 250 µl already combined OPD/substrate.
10. Shake rack.
11. Incubate 15 min. at room temperature in the dark.
12. Add 1 ml of stopping reagent per tube.
13. Read tubes at 492 nm in Resa COBAS spectrophotometer.
[0072] The assay results are shown in Tables 1 and 2.
TABLE 1
IMMUNOREACTIVITY OF RECOMBINANTLY EXPRESSED HTLV-I AND HIV-1 ENVELOPE PROTEINS (O.D.
= 492) |
SAMPLES |
NO. |
HIV-1 pENV(80) |
HIV-1 pENV(60)-HTLV-I-ENV-1 |
HIV-1 pENV(60)-HTLV-I-ENV-2 |
HIV-1 pENV(60)-HTLV-I-ENV-1 + ENV-2 |
HIV-1 POSITIVES |
1 |
1.134 |
1.304 |
0.959 |
2.459 |
|
2 |
4.471 |
3.157 |
3.137 |
4.918 |
|
3 |
4.224 |
3.211 |
2.957 |
4.582 |
|
4 |
3.981 |
4.650 |
2.998 |
4.600 |
|
5 |
4.422 |
3.395 |
3.263 |
4.858 |
HTLV-I POSITIVES |
1 |
0.102 |
1.983 |
0.690 |
3.772 |
|
2 |
0.120 |
1.287 |
1.350 |
3.135 |
|
3 |
0.074 |
0.685 |
1.071 |
2.757 |
|
4 |
0.075 |
1.860 |
0.700 |
3.608 |
|
5 |
0.143 |
2.604 |
0.300 |
4.577 |
NEGATIVES |
1 |
0.177 |
0.051 |
0.133 |
0.166 |
|
2 |
0.290 |
0.061 |
0.175 |
0.218 |
|
3 |
0.178 |
0.062 |
0.120 |
0.204 |
|
4 |
0.195 |
0.082 |
0.165 |
0.172 |
|
5 |
0.146 |
0.057 |
0.092 |
0.137 |
TABLE 2
DETECTION OF HUMAN ANTIBODY AGAINST HIV-1 AND HTLV-I USING RECOMBINANTLY EXPRESSED
ENVELOPE FUSION PROTEINS |
SAMPLES |
HIV-1 ASSAY |
HIV-1/HTLV-I COMBINATION ASSAY |
|
MEAN (O.D. 492) |
RANGE |
REACTIVE RATIO |
MEAN |
RANGE |
REACTIVE |
RATIO |
|
|
|
|
|
|
*HTLV-I POSITIVES (n=22) |
0.132 |
0.074-0.188 |
0/22 |
2.5 |
0.315-4.608 |
22/22 |
HIV-1 POSITIVES (n=100) |
3.980 |
1.134-5.206 |
100/100 |
4.576 |
2.586-5.691 |
100/100 |
NEGATIVES (n=100) |
0.195 |
0.096-0.396 |
0/100 |
0.194 |
0.053-0.338 |
0/100 |
*TEN SAMPLES WERE TESTED AT 1:100 DILUTION (INSTEAD OF 1:40) DUE TO INSUFFICIENT SAMPLE
VOLUME. |
[0073] While the invention has been described in connection with the preferred embodiment,
it is not intended to limit the scope of the invention to the particular form set
forth, but, on the contrary, it is intended to cover such alternatives, modifications,
and equivalents as may be included within the spirit and scope of the invention as
defined by the appended claims.
1. A hybrid gene comprising a first DNA sequence coding for a polypeptide containing
at least one epitope of the HIV-1 envelope protein which is recognized by antibodies
to HIV-1 and a second DNA sequence coding for a polypeptide containing at least one
epitope of the HTLV-I envelope protein which is recognized by antibodies to HTLV-I.
2. The gene of claim 1 wherein the first DNA sequence codes for a polypeptide containing
the immunodominant region of the HIV-1 gp41 transmembrane protein.
3 The gene of claim 2 wherein the DNA sequence codes for approximately amino acids
560-620 of the HIV-1 envelope protein.
4. The gene of claim 3 which contains a initiation DNA sequence operatively linked
to the hybrid gene.
5. The gene of claim 4 wherein the initiation DNA sequence is:
ATG AGA GGA TCC.
6. The gene of claim 5 wherein the second DNA sequence codes for a polypeptide containing
approximately amino acids 306-440 of the HTLV-1 envelope protein, said gene having
the nucleotide sequence
ATG AGA GGA TCC GAA GCT CAA CAG CAT CTG CTG CAA CTC ACT GTT TGG GGT ATC AAA CAG CTC
CAG GCT CGA ATT CTG GCT GTT GAA CGT TAC CTG AAA GAT CAA CAG CTC CTG GGT ATC TGG GGC
TGC AGT GGT AAA CTC ATC TGC ACT ACT GCT GTT CCT TGG AAT GCT TCT TGG TCT AAT AAG CTT
GGA TCC CGC TCC CGC CGA GCG GTA CCG GTG GCG GTC TGG CTT GTC TCC GCC CTG GCC ATG GGA
GCC GGA GTG GCT GGC GGG ATT ACC GGC TCC ATG TCC CTC GCC TCA GGA AAG AGC CTC CTA CAT
GAG GTG GAC AAA GAT ATT TCC CAG TTA ACT CAA GCA ATA GTC AAA AAC CAC AAA AAT CTA CTC
AAA ATT GCG CAG TAT GCT GCC CAG AAC AGA CGA GGC CTT GAT CTC CTG TTC TGG GAG CAA GGA
GGA TTA TGC AAA GCA TTA CAA GAA CAG TGC CGT TTT CCG AAT ATT ACC AAT TCC CAT GTC CCA
ATA CTA CAA GAA AGA CCC CCC CTT GAG AAT CGA GTC CTG ACT GGC TGG GGC CTT AAC TGG GAC
CTT GGC CTC TCA CAG TGG GCT CGA CCT GCA GCC AAG CTC AAG CTT GGC GAG ATT TTC AGG AGC
TAA
7. The gene of claim 5, wherein the second DNA sequence codes for a polypeptide containing
approximately amino acids 201-308 of the HTLV-I envelope protein, said gene having
the nucleotide sequence
ATG AGA GGA TCC GAA GCT CAA CAG CAT CTG CTG CAA CTC ACT GTT TGG GGT ATC AAA CAG CTC
CAG GCT CGA ATT CTG GCT GTT GAA CGT TAC CTG AAA GAT CAA CAG CTC CTG GGT ATC TGG GGC
TGC AGT GGT AAA CTC ATC TGC ACT ACT GCT GTT CCT TGG AAT GCT TCT TGG TCT AAT AAG CTT
GGA TCC GTC GAG CCC TCT ATA CCA TGG AAA TCA AAA CTC CTG ACC CTT GTC CAG TTA ACC CTA
CAA AGC ACT AAT TAT ACT TGC ATT GTC TGT ATC GAT CGT GCC AGC CTC TCC ACT TGG CAC GTC
CTA TAC TCT CCC AAC GTC TCT GTT CCA TCC TCT TCT TCT ACC CCC CTC CTT TAC CCA TCG TTA
GCG CTT CCA GCC CCC CAC CTG ACG TTA CCA TTT AAC TGG ACC CAC TGC TTT GAC CCC CAG ATT
CAA GCT ATA GTC TCC TCC CCC TGT CAT AAC TCC CTC ATC CTG CCC CCC TTT TCC TTG TCA CCT
GTT CCC ACC CTA GGA TCC AAG CTT GGC GAG ATT TTC AGG AGC TAA.
8. A hybrid polypeptide comprising a first amino acid subsequence containing at least
one epitope of the HIV-1 envelope protein which is recognized by antibodies to HIV-1,
and a second amino acid subsequence containing at least one epitope of the HTLV-I
envelope protein which is recognized by antibodies to HTLV-I.
9. The polypeptide of claim 8, wherein the first subsequence contains the immunodominant
region of the HIV-1 gp41 transmembrane protein.
10. The polypeptide of claim 9, wherein the first subsequence contains approximately
amino acids 560-620 of the HIV-1 envelope protein.
11. The polypeptide of claim 10 wherein the second amino acid subsequence contains
approximately amino acids 201-308 of the HTLV-I envelope protein, said polypeptide
having the amino acid sequence Met Arg Gly Ser Glu Ala Gln Gln His Leu Leu Gln Leu
Thr Val Trp Gly ILe Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp
Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp
Asn Ala Ser Trp Ser Asn Lys Leu Gly Ser Val Glu Pro Ser Ile Pro Trp Lys Ser Lys Leu
Leu Thr Leu Val Gln Leu Thr Leu Gln Ser Thr Asn Tyr Thr Cys Ile Val Cys Ile Asp Arg
Ala Ser Leu Ser Thr Trp His Val Leu Tyr Ser Pro Asn Val Ser Val Pro Ser Ser Ser Ser
Thr Pro Leu Leu Tyr Pro Ser Leu Ala Leu Pro Ala Pro His Leu Thr Leu Pro Phe Asn Trp
Thr His Cys Phe Asp Pro Gln Ile Gln Ala Ile Val Ser Ser Pro Cys His Asn Ser Leu Ile
Leu Pro Pro Phe Ser Leu Ser Pro Val Pro Thr Leu Gly Ser Lys Leu Gly Glu Ile Phe Arg
Ser.
12. The polypeptide of claim 10, wherein the second amino acid subsequence contains
approximately amino acids 306-440 of the HTLV-I envelope protein, said polypeptide
having the amino acid sequence
Met Arg Gly Ser Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly
Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Leu
Gly Ser Arg Ser Arg Arg Ala Val Pro Val Ala Val Trp Leu Val Ser Ala Leu Ala MET Gly
Ala Gly Val Ala Gly Gly Ile Thr Gly Ser MET Ser Leu Ala Ser Gly Lys Ser Leu Leu His
Glu Val Asp Lys Asp Ile Ser Gln Leu Thr Gln Ala Ile Val Lys Asn His Lys Asn Leu Leu
Lys Ile Ala Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu Asp Leu Leu Phe Trp Glu Gln Gly
Gly Leu Cys Lys Ala Leu Gln Glu Gln Cys Arg Phe Pro Asn Ile Thr Asn Ser His Val Pro
Ile Leu Gln Glu Arg Pro Pro Leu Glu Asn Arg Val Leu Thr Gly Trp Gly Leu Asn Trp Asp
Leu Gly Leu Ser Gln Trp Ala Arg Pro Ala Ala Lys Leu Lys Leu Gly Glu Ile Phe Arg Ser.
13. A recombinant vector comprising a hybrid gene which codes for a hybrid polypeptide
containing at least one epitope of the HIV-1 envelope protein which is recognized
by antibodies to HIV-1 and at least one epitope of the HTLV-I envelope protein which
is recognized by antibodies to HTLV-I.
14. The recombinant vector of claim 13 which is a plasmid capable of replication in
a unicellular host.
15. The recombinant vector of claim 14 which is capable of replication in an E. coli
strain.
16. The recombinant vector of claim 15 in which the vector is a member of the plasmid
pENV family.
17. The recombinant vector of claim 16 which is plasmid HIV-1 pENV(60)-HTLV-I-ENV-1.
18. The recombinant vector of claim 16 which is plasmid HIV-1 pENV(60)-HTLV-I-ENV-2.
19. A unicellular host containing a recombinant vector comprising a hybrid gene coding
for a hybrid polypeptide containing at least one epitope of the HIV-1 envelope protein
which is recognized by antibodies to HIV-1, and at least one epitope of the HTLV-I
envelope protein which is recognized by antibodies to HTLV-I.
20. The host of claim 19 which is an E. coli strain.
21. The E. coli strain of claim 20 which is E. coli M15.
22. A method for producing a hybrid polypeptide containing at least one epitope of
the HIV-1 envelope protein, and at least one epitope of the HTLV-I envelope protein
comprising culturing a unicellular host containing a recombinant vector which contains
a gene coding for said hybrid polypeptide under appropriate conditions of growth so
that said polypeptide is expressed and isolating said polypeptide.
23. A method for detecting antibodies to HTLV-I or HTLV-II envelope protein or HIV-1
envelope protein in mammalian body fluids comprising contacting a test sample with
a hybrid polypeptide containing at least one epitope of the HIV-1 envelope protein
and at least one epitope of the HTLV-1 envelope protein and allowing protein-antibody
complexes to form and detecting the complexes.
24. The method of claim 23 wherein the complexes are determined in an immunoassay.
25. The method of claim 24 which is an immunoassay selected from the group consisting
of Western Blotting, Double Antigen Sandwich Assay, Radioimmunoassay, or Enzyme Immunoassay.
26. The method of claim 25 comprising:
a) immobilizing a hybrid polypeptide of claims 8 to 12 on a solid support;
b) contacting a mammalian body fluid sample with the immobilized polypeptide of step
(a) and allowing the protein-antibody complex to form;
c) washing away unbound material from the complexes of step (b); and
d) detecting such complexes by the addition of labeled reagent capable of selectively
detecting human antibodies.
27. The method of claim 26 wherein the solid support is a bead.
28. The method of claim 27 wherein the reagent capable of selectively detecting human
antibodies is labeled anti-human IgG.
29. The method of claim 28 wherein the label is a radiolabel.
30. The method of claim 29 wherein the label is an enzyme label.
31. The method of claim 30 wherein any positive test result is confirmed by testing
the sample separately for antibodies to HTLV-I or HIV-1 envelope protein
32. The use of a hybrid protein of claims 8 to 12 for testing mammalien body fluids
for the presence of antibodies to HTLV-I, HTLV-II, or HIV-1 envelope proteins.
33. A diagnostic kit useful for measuring antibodies to HTLV-1, HTLV-II, or HIV-1
envelope protein in mammalian body fluids comprising:
a) a container with a hybrid protein of claims 8 to 12 immobilized on a solid support;
b) a container with labeled reagent capable of selectively detecting human antibodies;
c) containers with positive and negative controls; and
d) a container with sample diluent.
34.The test kit of claim 33 wherein the solid support is a bead.